Technical Note Tissue Section Imaging Published August 2017. The most recent version of this Technical Note is posted at licor.com/bio/support.
Page 2 - Tissue Section Imaging Table of Contents Page I. Tissue Section Imaging and Microscopy 2 II. Fluorescence-based Tissue Analysis Procedure 3 III. Tissue Preparation 4 Fixation 4 Fixatives Options 4 Fixating Factors 4 Dehydration 5 IV. Preserve Tissue 5 Paraffin-embedded 5 Frozen Section 6 Free Floating Section 6 V. Cut Tissue Sections 6 VI. Remove Paraffin or Optimal Cooling Temperature Compound 7 Remove Paraffin 7 Remove Optimal Cutting Temperature Compound 7 VII. Retrieve Epitope 7 VIII. Permeabilize 8 IX. Block 8 X. Mount 8 XI. Image Tissue Section 9 XII. Imaging Tissue Sections Resources 9 I. Tissue Section Imaging and Microscopy Tissue section imaging and microscopy are effective for granular identification of targeting agent locations within an organ. With appropriate microscopy equipment, you can assess NIR-labeled targeting agent location (e.g. membrane bound, intracellular, or interstitial).
Tissue Section Imaging - Page 3 However, most microscopes are not configured for NIR fluorescence detection. If you're ready to try IRDye Infrared Dyes on an epifluorescence microscope, this list of microscope requirements will help you get started (licor.com/microscope_requirements). The following procedure outlines the basic steps required to prepare tissue sections for imaging on the Odyssey CLx or for examination under a microscope. More Info: You can also send your samples to LI-COR Custom Services for imaging with the Odyssey CLx or the Olympus IX81 microscope (licor.com/customservices). II. Fluorescence-based Tissue Analysis Procedure Some testing and optimization will be necessary to fine-tune this general protocol for your specific experiment. Research the current literature from antibody vendors to quickly develop the best procedure (references are provided at the end of this procedure). Note: Some post-processing steps, ie steps performed after the tissue section has been cut, can deplete NIR fluorescent signal from an in vivo administered optical probe in the tissue (especially the paraffin removal and epitope retrieval steps). If you plan to use immunofluorescent procedures after cutting a tissue section, be sure to document the tissue section signal before post-processing. If you plan to use antibodies, carefully select and validate primary and secondary antibodies. Try to find antibodies that have been validated for immunohistochemistry. Antibodies that have been validated for use in Western blot applications need to also be evaluated in tissue to ensure the antibody specificity within the tissue. 1. Prepare tissue 2. Preserve tissue 3. Cut tissue sections 4. Remove paraffin or optimal cooling temperature compound (OCT) 5. Retrieve epitope, if necessary 6. Permeabilize
Page 4 - Tissue Section Imaging 7. Block 8. Mount 9. Image III. Tissue Preparation Begin by fixing and dehydrating the sample. Fixation The goal of fixation is to preserve the tissue s cellular structure as closely as possible to its native state. If fixation is done correctly, the sample can be used throughout the rest of the analysis without losing the protein of interest. Fixation may damage or mask antigenic sites, potentially compromising the intensity of your immunostaining. You may need to test several fixatives to determine which fixative adequately preserves both effective antibody binding and structural integrity of the sample. Fixatives Options There are two common types of chemical fixatives: Cross-linking fixatives: These fixatives act by creating covalent bonds between proteins in the tissue. The most common fixative in histology is formaldehyde gas dissolved in water (37% w/v), but other fixatives can be used. o o Formalin is a formaldehyde solution with methanol added to prevent formaldehyde polymerization. Typically, formalin is used as a 10% solution in neutral buffer. Paraformaldehyde is a polymerized form of formaldehyde, which may be better for use with some antibodies than formaldehyde. Precipitating fixatives: Alcohols are commonly used to fix frozen sections and smears. A variety of factors affect fixation. Some fixatives, if present in tissue, can diminish intended dye/fluorophore signal. This problem can occur when a labeled biomarker is used in vivo prior to tissue excision. Fixating Factors The following factors are important to consider as you plan to fix your samples.
Tissue Section Imaging - Page 5 ph: The fixative should be buffered to between 7.2-7.4. Osmolarity: Formalin is typically used as a 10% solution in neutral buffer. Specimen size: Typically, samples should be 4-10 µm thick. Fixative volume: The volume of fixative should generally be 15-20 times greater than that of the tissue section. Temperature: Tissues are usually fixed at room temperature. Duration: The rule of thumb is to treat the tissue with fixative for 1 hour for every 1mm of tissue thickness. Dehydration You must dehydrate tissues prior to paraffin embedding, because paraffin is immiscible with water. Dehydration is accomplished by displacing water in the tissue through a series of alcohol baths containing increasing concentrations of alcohol, usually 30%, 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 2 hours each. A second treatment of 100% alcohol is used to ensure that all water has been removed from the sample. Important: Never allow tissues to air dry. IV. Preserve Tissue You have many options for tissue preservation. The most common methods are described below. Paraffin-embedded Wax preserves tissue morphology and can be used to preserve tissues for years at room temperature. The most commonly used waxes are commercial paraffin waxes. Tissue processors will likely have instrument specific procedures for paraffin embedding. The following steps show the general procedure for paraffin embedding. 1. 70% ethanol treatment 2. 95% ethanol treatment 3. Four treatments with 100% ethanol
Page 6 - Tissue Section Imaging 4. Two treatments with a clearing agent (such as Xylene) 5. Two treatments with wax at 58 C Frozen Section The Frozen Tissue section method allows you to preserve enzyme and antigen function in your sample. The tissue section must be kept thin so it will freeze quickly before ice crystals form, which can cause artifacts in the sample. Properly preserved specimens can be kept for about a year at -80 C. Optimal cutting temperature compounds, made of water-soluble glycols and resins, are often used as a convenient specimen matrix for cryostat sectioning of tissues at temperatures of -10 C and below. Tissues are usually sectioned into 2cm cubed pieces. Free Floating Section Free-floating immunochemistry is performed with sections floating in solution, usually in a large well plate. Sections are not mounted on slides until after the immunochemistry process has been completed. Free-floating immunohistochemistry is ideal for fixed tissues. This method can be used on thicker sections, making it practical for seeing distribution of staining through the entire section. More Info: This example paper shows the use of free floating tissue sections (licor.com/brain_sections). V. Cut Tissue Sections For microscopy, sections are generally cut to a thickness of 4-10 µm. Thicker sections may facilitate visualization of lower-abundance protein targets when imaging tissues on the Odyssey CLx. The following tools are commonly used to cut tissue sections. Microtome: Tool used for cutting thin slices of tissue for microscopy. Cryostat: Tool used to cut frozen sections.
Tissue Section Imaging - Page 7 VI. Remove Paraffin or Optimal Cooling Temperature Compound Remove Paraffin The following general procedure allows you to remove paraffin and rehydrate the tissue. 1. Two treatments with Xylene 2. Two treatments with 100% ethanol 3. 95% ethanol 4. 70% ethanol 5. 50% ethanol 6. Deionized water rinse 7. Rehydrate with wash buffer Remove Optimal Cutting Temperature Compound If you have used OCT to preserve your tissue, you do not necessarily need to remove it before imaging. Consult the manufacturer's instructions for more information. This is the general procedure for removing OCT. 1. Absolute acetone treatment for 10-15 seconds 2. 95% ethanol treatment for 10-15 seconds 3. Absolute acetone for 10 seconds 4. 95% ethanol for 10 seconds VII. Retrieve Epitope Fixation can alter protein biochemistry, causing the epitope of interest to be masked from the primary antibody. The epitope can be masked by cross-linking of amino acids within the epitope or cross-linking with unrelated peptides at or near the epitope. The process of epitope/antigen retrieval refers to unmasking the epitope to restore epitope/antibody binding. The epitope can be unmasked in several ways: Acid, basic, neutral treatment Heat Microwaves
Page 8 - Tissue Section Imaging VIII. Permeabilize Permeabilization helps get antibodies into fixed tissue samples. The extent of permeabilization required depends on the target. If you are detecting a cell surface protein, little, if any, permeabilization will be required. If you do permeabilize, be careful not to remove the entire membrane. Permeabilization, like fixation, can affect the morphology of cells, which will affect the antigen of interest. Commonly used detergents include NP-40, Triton X-100, Tween-20, and Saponin. Important: Optimize your procedure for each target of interest. IX. Block The staining procedure can be improved by blocking the non-specific binding of your antibody to the tissue section. BSA, milk, or serum can be used for this. Important: Be careful not to block with BSA from the same species as the species in which the primary antibody was raised! The blocking procedure should be optimized prior to examining valuable tissue samples. X. Mount When mounting sample on a slide, it is important to use low fluorescence mounting media. Low fluorescence mounting media are available from several vendors. Important: Some mounting media have fluorescent properties and are visible in either the 700 nm or 800 nm channel. Test your mounting medium before using it on valuable samples!
Tissue Section Imaging - Page 9 XI. Image Tissue Section Finally, tissues can be imaged using microscopy techniques or whole tissue sections can be imaged on the Odyssey CLx Imager. XII. Imaging Tissue Sections Resources "Immunofluorescence General Protocol." Cell Signaling Technology. Web. 21 July 2017.cellsignal.com/contents/resources-protocols/immunofluorescence-general-protocol/if "Antibody Validation for Immunofluorescence." Cell Signaling Technologies.cellsignal.com/common/content/content.jsp?id=ourApproach-validation-if "Protocol for the Preparation and Fluorescent IHC Staining of Frozen Tissue Sections." R&D Systems. Web. 21 July 2017. rndsystems.com/resources/protocols/protocol-preparationand-fluorescent-ihc-staining-frozen-tissue-sections "Double immunofluorescence simultaneous protocol." Abcam. Web. 21 July 2017abcam.com/ps/pdf/protocols/double%20immunofluorescence%20- simultaneous%20protocol.pdf "IHC staining protocol for paraffin, frozen and free floating sections." Abcam. Web. 21 July 2017. abcam.com/protocols/immunostaining-paraffin-frozen-free-floating-protocol "Free-Floating Versus Slide-Mounted Sections for Immunohistochemistry." Bitesize Bio. Web. 21 July 2017. bitesizebio.com/26549/free-floating-versus-slide-mounted-sections-ihc
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